CN101148680A - Method for leading plasmid carrier containing gene cure segment in cell by nano particles - Google Patents
Method for leading plasmid carrier containing gene cure segment in cell by nano particles Download PDFInfo
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- CN101148680A CN101148680A CNA2007101338655A CN200710133865A CN101148680A CN 101148680 A CN101148680 A CN 101148680A CN A2007101338655 A CNA2007101338655 A CN A2007101338655A CN 200710133865 A CN200710133865 A CN 200710133865A CN 101148680 A CN101148680 A CN 101148680A
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Abstract
The present invention relates to process of leading plasmid carrier containing gene therapy segment into cell with nanometer particle. The process includes the following steps: inoculating cell in good growth condition to cell culturing plate and culturing; centrifuging nanometer inorganic salt suspension and dissolving the precipitate in deionized water in 500 microliter; dissolving 2 microliter of plasmid in 98 microliter of deionized water; compounding 1 ml of suspension with solution of nanometer particle in 5-10 mg and solution of plasmid in 4-15 microliter through shaking for best combination between the nanometer particle and the plasmid; centrifuging the suspension to obtain the precipitate, adding deionized water, adding to the cell culturing plate, and adding fresh culture liquid to culture for the nanometer particle well combined to the plasmid to transfect the cell; and finally observing the transfection effect in a microscope.
Description
Technical field
The present invention relates to a kind of method that will contain the plasmid vector transfered cell of gene therapy segment, especially a kind of method that will contain the plasmid vector transfered cell of gene therapy segment by nanoparticle.
Background technology
(RNA interference RNAi) is a kind of " gene silencing " that is brought out by double-stranded RNA in the RNA interference.In this process, there is the messenger RNA(mRNA) of homologous sequence to be degraded with double-stranded RNA in the pathogenic cell, thereby suppressed the expression of Disease-causing gene, the RNA perturbation technique is being detected gene function and treatment has broad application prospects aspect the human diseases.SiRNA is the disconnected double stranded rna molecule of a kind of RNA of being used for interferential short-movie, can be the specific mRNA of target degraded with the mRNA of homology complementary sequence, can be used as a kind of gene therapy medicament with target.
The siRNA medicine comprises the siRNA of chemosynthesis, the carrier of expression siRNA (expressing the plasmid of siRNA, the virus of expression siRNA).The carrier of expressing siRNAs contains rna plymerase iii promotor or rna plymerase ii promoter sequence usually, at first transcribe and generate the shRNA similar, in cell, become siRNA performance gene silencing effect then to the miRNA precursor. in vivo with the cultured tissue cell in use expression siRNA carrier can be incorporated into for a long time and also " knock out " endogenous gene in the genome.Make siRNA successfully be applied to the treatment of cancer and transmissible disease, the siRNA medicine must possess following condition: enough little, can pass cytolemma, and enter cell; Not by intracellular nuclease degradation; Can arrive the suitable position of cell; In cell, can correctly fold after the connection carrier; Can return specific target cell, as scavenger cell, T cell, hemopoietic stem cell, thus the degraded or the drug effect of realization carrier.Therefore, developing system that a kind of safe, effective tool specificity has no adverse reaction again as siRNA medicine import system, is a urgent problem.
At present, adopt micro-injection, electroporation, liposome method, infusion method, engineering bacteria to feed method, transgenosis method and virus infection method the siRNA medicine is imported in the mammalian cell usually.Up to now, the most effective introduction method is to utilize the viral vector infection method of expressing siRNA, but because the limitation of viral system, as security, tumorigenicity and immunogenicity, limited DNA loading capacity, carrier assembling difficulty is big, spends high shortcoming and makes its application be subjected to considerable restraint.For example, on September 13rd, 1999, the U.S. human gene therapy of University of Pennsylvania institute utilizes adenovirus to make carrier 18 years old patient Gelsinger is done ornithine carbamyl transferase gene therapy patient's death after .4 days. and the cause of death is that the immune response that adenovirus causes causes hepatitis gravis, multiple organ dysfunction syndrome, these have all hindered clinical application of gene therapy.
Along with the develop rapidly of nanometer biotechnology, based on the non-virus carrier system of nano material---new vitality has been injected in the gene therapy that appears as of nano material carrier system.Utilize nano material to carry the plasmid vector of expressing siRNA and enter mammalian cell, have following advantage: reduced immunogenicity; In very little volume, also can store relatively large gene segment; Can good provide protection be arranged to the dna segment that inserts wherein.
(Human telomerase reverse transcriptase, hTERT) gene is the desirable molecular target of oncotherapy to human telomerase reverse transcriptase.HTERT is the important component part of Telomerase complex body, in 80%～90% tumour cell, express, and and telomerase activation between high correlation is arranged, be the rate-limiting factor of telomerase activation.HTERT siRNA expression vector has the effect that suppresses tumor cell proliferation and promote apoptosis of tumor cells, there has been bibliographical information hTERT siRNA expression vector can suppress HeLa Cells at present, transitional cell bladder carcinoma cell line, liver cancer cell, stomach cancer cell, the propagation of tumour cells such as nasopharyngeal carcinoma cell, can promote the HeLa cell, the apoptosis of tumour cells such as tongue squamous cell carcinoma, transfection method comprises electroporation, liposome method, polyamide-amide type dendroid superpolymer (G5 PAMAM-D) Nano microsphere mediated method etc. studies show that the Nano microsphere mediated method is better than liposome method to the effect of hTERT mRNA expression inhibiting.
The CN100335038C patent documentation discloses a kind of preparation method who contains the inorganic salt nano particles that biologically active substance and traditional Chinese medicine ingredients seal, and still, can this inorganic salt nano particles mediate the plasmid transfection cell, do not make reports.
Summary of the invention
The objective of the invention is to: at present therapeutic gene effectively being transported to the practical problems that the conventional carriers in the target cell exists in practice, select a kind of new carrier, and a kind of method that will be contained the plasmid vector transfered cell of gene therapy segment by nanometer is provided.
The object of the present invention is achieved like this: a kind ofly will contain the method for the plasmid vector transfered cell of gene therapy segment by nanoparticle, and it is characterized in that:
A) growth conditions good cell, cultivation overnight, 1X10 is got in 0.25% trysinization after the cell counting
6Cell inoculation 12 orifice plates, add fresh culture DMEM, at 37 ℃, 5%CO
2Cultivate under the condition, change liquid once after 24 hours, 24h is re-seeded into each hole of Tissue Culture Plate in 12 holes with an amount of cell before the transfection, and attached cell carries out transfection when covering with floorage 80%;
B) get the suspension that the inorganic salt nano particles net content is 20～30mg, in 4 degrees centigrade, centrifugal 15 minutes of 8500rpm abandons supernatant, and precipitation is dissolved in the 500ul deionized water, is distributed into 5 parts, every part of 120ul;
C) draw the 2ul plasmid and be dissolved in the 98ul deionized water, spectrophotometric measures plasmid vector concentration 1084ug/ml, purity 1.65;
D) with b) nanoparticle content that obtains in the step is solution and the c of 5～10mg) the plasmid content that obtains in the step is that the solution of 4～15ug is made into the suspension that cumulative volume is lml, in 4 degrees centigrade, 96rpm shook up in 4 hours, and nanoparticle is fully combined with plasmid;
E) get nanoparticle suspension lml after shaking up in 4 degrees centigrade, 8500rpm, centrifugal 15 minutes, abandon supernatant, add 200ul deionized water, mixing in the precipitation, evenly add 4 holes in the Tissue Culture Plate in 12 holes, jog makes it to mix, and adds the 2ml fresh medium again in every hole, at 37 ℃, 5%CO
2Incubated overnight under the condition makes with the well-bound nanoparticle of plasmid and is transfected into cell;
F) observe in microscopically after 24 hours, the plasmid vector that contains gene therapy segment is imported into cell.
In technical scheme of the present invention: described cell is a glioma cell; Described plasmid is the plasmid vector that contains gene therapy segment, wherein: gene therapy segment is taken from human telomerase reverse transcriptase (hTERT) gene of No.NM003219 among the GenBank, and the plasmid vector that contains gene therapy segment is meant human telomerase reverse transcriptase (hTERT) gene is inserted plasmid vector pGCsi-H1/neo/GFP; Described being meant by laser confocal microscope or fluorescence inverted microscope in the microscopically observation observed.
In technical scheme of the present invention: contain 10% foetal calf serum among the described fresh culture DMEM.
In technical scheme of the present invention: described inorganic salt nano particles is the inorganic salt nano particles that traditional Chinese medicine ingredients is sealed.
In technical scheme of the present invention: the inorganic salt nano particles that described traditional Chinese medicine ingredients is sealed is the inorganic salt nano particles that astragalus polysaccharides is sealed composition.
The invention has the advantages that: comparing with the virus vector in the up to now efficient gene transfer method at present based on the non-virus carrier system of nanoparticle has following advantage: nontoxicity, non-immunogenicity, increase the transfection efficiency of cell to gene, in very little volume, also can store relatively large gene segment, can good provide protection be arranged the dna segment that inserts wherein; Inorganic salt nano particles is biological degradation in vivo, and the inorganic salt nano particles of sealing the astragalus polysaccharides composition can improve the physicochemical property of inorganic salt nano particles, improves the ability of carrying gene segment, and the dna segment that inserts is wherein had good provide protection.
Description of drawings
Figure l is the good glioma U251 cell of growth conditions that microscopically is observed;
Fig. 2 is the situation behind people's glioma cell U251 transfection nanoparticle of observing under the laser confocal microscope;
Fig. 3 is the situation behind people's glioma cell U251 transfection nanoparticle of observing under the fluorescence inverted microscope;
Fig. 4 is the electrophorogram relevant with effect of the present invention.
Embodiment
Embodiment 1:
The inorganic salt nano particles that the preparation traditional Chinese medicine ingredients is sealed
Preparation before the preparation
Salts solution: with the Manganous chloride tetrahydrate of 0.01～1.0 mole of deionized water preparation (or magnesium chloride, or calcium chloride solution).
Phosphoric acid solution: with the phosphoric acid solution of 0.01～1.0 mole of deionized water preparation.
The active substance solution of Chinese medicine: prepare the active substance solution of Chinese medicine astragalus polysaccharide in the every 100ml physiological saline+ratio of 1mg astragalus polysaccharides.
PH value is that 7.0,0.01 moles phosphoric acid buffer can outsourcing.
Preparation process is as follows:
A) preparation inorganic salt nano particles:
With 0.01～1.0 mole salts solution, 0.01～1.0 mole phosphoric acid solution by mix at 1: 1 place agitator 7 after, stirred 48 hours down at 4 ℃, passed through ultrasonic disruption instrument 9 supersound process 15 minutes again, then under 4 ℃, is centrifugal 15 minutes of 8500rpm through supercentrifuge 8 at rotating speed, removes supernatant liquor, uses the deionized water suspended particles, concentration at 5mg/ml forms the inorganic salt particle, and its size dimension is 100nm～800nm;
B) seal the active substance of Chinese medicine:
Under the room temperature, after the active substance (present embodiment is the active substance solution of Chinese medicine astragalus polysaccharide) of inorganic salt nano particles, Chinese medicine mixed by 1: 10, concentration by 10mg/ml is suspended in pH value 7.0,0.01 in the phosphoric acid buffer of mole, shaking on shaking table 10 2 hours under 4 ℃, under 4 ℃, is centrifugal three times of 8500rpm through supercentrifuge 8 at rotating speed then, each 15 minutes, obtain the inorganic salt nano particles that traditional Chinese medicine ingredients is sealed.
The inorganic salt nano particles that the traditional Chinese medicine ingredients (astragalus polysaccharides) that obtains is sealed should be stored in 4 ℃ or by the freeze drier freeze-dried back.
Embodiment 2
The method that will contain the plasmid vector transfered cell of gene therapy segment by nanoparticle
Preparation before the preparation
1, cell: human glioma cell U251 is available from cell resource center of Shanghai Sheng Ke institute.
2, plasmid: the plasmid vector that contains gene therapy segment, be that human telomerase reverse transcriptase (hTERT) gene is inserted plasmid vector pGCsi-H1/neo/GFP, wherein: gene therapy segment is taken from human telomerase reverse transcriptase (hTERT) gene of No.NM 003219 among the GenBank, can obtain by the human gene bank; Plasmid vector pGCsi-H1/neo/GFP is provided by Chinese Ji Kai company.
3, inorganic salt nano particles is according to the method preparation of embodiment 1.
Preparation process
A) the human glioma cell U251 that growth conditions is good, cultivation overnight, 1X10 is got in 0.25% trysinization after the cell counting
6Cell inoculation 12 orifice plates, add fresh culture DMEM (containing 10% foetal calf serum among the DMEM), at 37 ℃, 5%CO
2Cultivate under the condition, change liquid once after 24 hours, 24h is re-seeded into each hole of Tissue Culture Plate in 12 holes with cell U251 before the transfection, and attached cell U251 carries out transfection when covering with floorage 80%;
B) get the suspension that the inorganic salt nano particles net content is 20～30mg, in 4 degrees centigrade, centrifugal 15 minutes of 8500rpm abandons supernatant, and precipitation is dissolved in the 500ul deionized water, is distributed into 5 parts, every part of 120ul;
C) draw the 2ul plasmid and be dissolved in the 98ul deionized water, spectrophotometric measures plasmid vector concentration 1084ug/ml, purity 1.65;
D) with b) nanoparticle content that obtains in the step is solution and the c of 5～10mg) the plasmid content that obtains in the step is that the solution of 4～15ug is made into the suspension that cumulative volume is 1ml, in 4 degrees centigrade, 96rpm shook up in 4 hours, and nanoparticle is fully combined with plasmid;
E) get nanoparticle suspension 1ml after shaking up in 4 degrees centigrade, 8500rpm, centrifugal 15 minutes, abandon supernatant, add the 200ul deionized water in the precipitation, mixing, evenly add 4 holes in the Tissue Culture Plate in 12 holes, jog makes it to mix, and all the other each holes in contrast, in every hole, add the 2mi fresh medium again, at 37 ℃, 5%CO
2Incubated overnight under the condition makes with the well-bound nanoparticle of plasmid and is transfected into cell;
F) observe in microscopically after 24 hours, contain the plasmid vector transfered cell of gene therapy segment.
D at present embodiment) in the step, with b) nanoparticle content that obtains in the step is respectively solution and the c of 1mg, 5mg, 10mg) the plasmid content that obtains in the step solution that is respectively 5ug, 10ug, 20ug is made into the suspension that cumulative volume is 1ml, in 4 degrees centigrade, 96rpm, shook up in 4 hours, nanoparticle is fully combined with plasmid, and its test-results sees Table 1
Table 1 nanoparticle and plasmid adopt the encapsulation rate of different mixing proportion
By table 1 as seen, when the blending ratio that adopts 10 milligrams of nanoparticles and 10 microgram plasmids is made into the suspension that cumulative volume is 1ml, its encapsulation rate can reach 98.3%.
Embodiment 3
The effect that will contain the plasmid vector transfered cell of gene therapy segment by nanoparticle:
Fig. 1 is the good glioma cell U251 of growth conditions that takes from contrast, by the observations of fluorescence inverted microscope (Nikon Eclipse TE 2000-S).
Fig. 2 as seen, after the mode transfection of employing embodiment 2 is finished, the cover glass that covers with cell is placed laser confocal microscope (ZEISS LSM510,40 * (magnification)) situation under behind observer's glioma cell U251 transfection nanoparticle, under laser confocal microscope, can see, glioma cell growth conditions behind the transfection nanoparticle good (as the arrow indication), wherein green portion is that transfection success back GFP (green fluorescent protein) expresses the green fluorescence (seeing whiting place of artwork master arrow front end) that produces.
Fig. 3 as seen, after the mode transfection of employing embodiment 2 is finished, the cover glass that covers with cell is placed the situation behind observer's glioma cell U251 transfection nanoparticle under the fluorescence inverted microscope (Nikon Eclipse TE 2000-S), under the fluorescence inverted microscope, can see, behind the transfection nanoparticle (as the arrow indication), transfection success back GFP expresses the same green fluorescence (seeing whiting place of artwork master arrow front end) that produces.
Fig. 4 carries out the process that gene is sealed to the present invention, judges the influence of the present invention to the 26S Proteasome Structure and Function of gene by electrophorogram, among the figure: the 1st electrophoresis road: gene standard substance; The 2nd electrophoresis road: the plasmid that the nanoparticle of sealing from plasmid decomposes; The 3rd electrophoresis road: not with nanometer bonded plasmid.As seen from Figure 4, the 2nd and the shown gene band in the 3 two electrophoresis road identical on quantity and position, illustrate that the gene that is encapsulated on the nanometer can not be degraded and loss of function because of encapsulation process.
The transfection efficiency that will be contained the plasmid vector transfered cell of gene therapy segment by nanoparticle can reach 86%, though this transfection efficiency is lower than the transfection efficiency of viral vector infection method, really will exceed nearly about one times than present business-like liposome.
Adopt the viral vector infection method to have its limitation: at first to be its safety issue, there are many potential danger in virus vector for the curer, as cytotoxicity, immunogenicity and inflammatory reaction are used the viral vector infection method and the danger of inserting mutagenesis and causing tumour also can be occurred; Secondly, in the viral vector infection method, have only the DNA of small segment can be inserted in the viral genome, big segmental DNA then is difficult to insert wherein.And the present invention does not exist this type of risk and hidden danger, and the plasmid vector transfered cell that nanoparticle will contain gene therapy segment has very big advantage.
More than each embodiment be not to concrete restriction of the present invention.
Claims (5)
1. one kind will contain the method for the plasmid vector transfered cell of gene therapy segment by nanoparticle, it is characterized in that:
A) growth conditions good cell, cultivation overnight, 1X10 is got in 0.25% trysinization after the cell counting
6Cell inoculation 12 orifice plates, add fresh culture DMEM, at 37 ℃, 5%CO
2Cultivate under the condition, change liquid once after 24 hours, 24h is re-seeded into each hole of Tissue Culture Plate in 12 holes with an amount of cell before the transfection, and attached cell carries out transfection when covering with floorage 80%;
B) get the suspension that the inorganic salt nano particles net content is 20～30mg, in 4 degrees centigrade, centrifugal 15 minutes of 8500rpm abandons supernatant, and precipitation is dissolved in the 500ul deionized water, is distributed into 5 parts, every part of 120ul;
C) draw the 2ul plasmid and be dissolved in the 98ul deionized water, spectrophotometric measures plasmid vector concentration 1084ug/ml, purity 1.65;
D) with b) nanoparticle content that obtains in the step is solution and the c of 5～10mg) the plasmid content that obtains in the step is that the solution of 4～15ug is made into the suspension that cumulative volume is 1ml, in 4 degrees centigrade, 96rpm shook up in 4 hours, and nanoparticle is fully combined with plasmid;
E) get nanoparticle suspension 1ml after shaking up in 4 degrees centigrade, 8500rpm, centrifugal 15 minutes, abandon supernatant, add 200ul deionized water, mixing in the precipitation, evenly add 4 holes in the Tissue Culture Plate in 12 holes, jog makes it to mix, and adds the 2ml fresh medium again in every hole, at 37 ℃, 5%CO
2Incubated overnight under the condition makes with the well-bound nanoparticle of plasmid and is transfected into cell;
F) observe in microscopically after 24 hours, the plasmid vector that contains gene therapy segment is imported into cell.
2. according to claim 1ly will contain the method for the plasmid vector transfered cell of gene therapy segment by nanoparticle, it is characterized in that: described cell is a glioma cell; Described plasmid is the plasmid vector that contains gene therapy segment, wherein: gene therapy segment is taken from human telomerase reverse transcriptase (hTERT) gene of No.NM 003219 among the GenBank, and the plasmid vector that contains gene therapy segment is meant human telomerase reverse transcriptase (hTERT) gene is inserted plasmid vector pGCsi-H1/neo/GFP; Described being meant by laser confocal microscope or fluorescence inverted microscope in the microscopically observation observed.
3. according to claim 1ly will contain the method for the plasmid vector transfered cell of gene therapy segment, it is characterized in that: contain 10% foetal calf serum among the described fresh culture DMEM by nanoparticle.
4. according to claim 1ly will contain the method for the plasmid vector transfered cell of gene therapy segment by nanoparticle, it is characterized in that: described inorganic salt nano particles is the inorganic salt nano particles that traditional Chinese medicine ingredients is sealed.
5. according to claim 4ly will contain the method for the plasmid vector transfered cell of gene therapy segment by nanoparticle, it is characterized in that: the inorganic salt nano particles that described traditional Chinese medicine ingredients is sealed is the inorganic salt nano particles that astragalus polysaccharides is sealed.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820492A (en) * | 2013-10-29 | 2014-05-28 | 王深明 | Functional nano catechin gene-introduction material and preparation method thereof |
| CN104471063A (en) * | 2012-06-20 | 2015-03-25 | 独立行政法人科学技术振兴机构 | Nucleic acid complex and nucleic acid-polysaccharide complex |
| CN114292753A (en) * | 2021-12-23 | 2022-04-08 | 南京大学 | Method for separating cells and discharging nanoparticles from cells |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335038C (en) * | 2005-07-25 | 2007-09-05 | 江桥 | Preparation process and application of inorganic salt nanometer particle containing biological active material and enveloped Chinese medicinal ingredient |
| WO2007067733A2 (en) * | 2005-12-09 | 2007-06-14 | Massachusetts Institute Of Technology | Compositions and methods to monitor rna delivery to cells |
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2007
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104471063A (en) * | 2012-06-20 | 2015-03-25 | 独立行政法人科学技术振兴机构 | Nucleic acid complex and nucleic acid-polysaccharide complex |
| CN103820492A (en) * | 2013-10-29 | 2014-05-28 | 王深明 | Functional nano catechin gene-introduction material and preparation method thereof |
| CN114292753A (en) * | 2021-12-23 | 2022-04-08 | 南京大学 | Method for separating cells and discharging nanoparticles from cells |
| CN114292753B (en) * | 2021-12-23 | 2023-06-16 | 南京大学 | Method for separating cells and discharging nanoparticles from cells |
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